Endoplasmic reticulum (ER) stress represents an early pathological event in amyotrophic lateral sclerosis (ALS). ATF4 is a key ER stress transcription factor that plays a role in both adaptation to stress and the activation of apoptosis. Here we investigated the contribution of ATF4 to ALS. ATF4 deficiency reduced the rate of birth of SOD1G86R transgenic mice. The fraction of ATF4−/−-SOD1G85R transgenic mice that were born are more resistant to develop ALS, leading to delayed disease onset and prolonged life span. ATF4 deficiency completely attenuated the induction of pro-apoptotic genes, including BIM and CHOP, and also led to quantitative changes in the ER protein homeostasis network. Unexpectedly, ATF4 deficiency enhanced mutant SOD1 aggregation at the end stage of the disease. Studies in the motoneuron cell line NSC34 demonstrated that knocking down ATF4 enhances mutant SOD1 aggregation possibly due to alteration in the redox status of the cell. Our results support a functional role of ATF4 in ALS, offering a novel target for disease intervention.
Huntington disease (HD) is caused by an extended polyglutamine [poly(Q)] stretch in the Huntingtin (HTT) protein, and is associated with the accumulation of intracellular protein aggregates, onset of progressive chorea, psychiatric symptoms and dementia. Although the mechanism underlying the pathological effects of mutant HTT (mHTT) remains highly controversial, accumulating evidence suggest that protein-folding stress at the endoplasmic reticulum (ER) may contribute to mHTT-mediated degeneration. ER stress is alleviated by the activation of an adaptive reaction known as the unfolded protein response (UPR), whereas chronic ER stress triggers apoptosis by the same pathway. However, most of the studies linking ER stress with HD in vivo are correlative. UPR signaling is initiated by the activation of at least three distinct stress sensors located at the ER membrane known as ERN1/IRE1α, EIF2AK3/PERK and ATF6. These stress sensors control the expression of specialized transcription factors that modulate the upregulation of a variety of target genes involved in folding, protein quality control, autophagy and protein synthesis.
Huntington disease; autophagy; XBP1; ER stress; aging; neurodegeneration
Protein folding stress is a salient feature of the most frequent neurodegenerative diseases. Although the accumulation of abnormally folded proteins is a well-characterized event underlying the pathology, the way cells respond to this phenomenon is not well understood. Signs of endoplasmic reticulum (ER) stress are a common marker of neurodegeneration in many diseases, which may represent two contrasting processes: cell protection events due to activation of adaptive programs, or a chronic stress state that culminates in apoptosis to eliminate irreversibly injured cells. Autophagy has been proposed as a protective mechanism to overcome neurodegeneration that is also modulated by ER stress. In this issue of autophagy Bertrand Mollereau’s group provides novel evidence indicating that engagement of nonharmful levels of ER stress protects against experimental Parkinson disease. At the mechanistic level, a homeostatic crosstalk between ER stress signaling and the autophagy pathway was proposed to mediate the therapeutic effects. This study, together with recent findings, supports the involvement of a “hormesis mechanism” to handle degeneration through preconditioning mediated by a dynamic balance between ER stress and autophagy. The implications for aging and future therapeutic development are discussed.
autophagy; ER stress; Parkinson disease; neurodegeneration; protein misfolding
Mutations leading to expansion of a poly-glutamine track in Huntingtin (Htt) cause Huntington's disease (HD). Signs of endoplasmic reticulum (ER) stress have been recently reported in animal models of HD, associated with the activation of the unfolded protein response (UPR). Here we have investigated the functional contribution of ER stress to HD by targeting the expression of two main UPR transcription factors, XBP1 and ATF4 (activating transcription factor 4), in full-length mutant Huntingtin (mHtt) transgenic mice. XBP1-deficient mice were more resistant to developing disease features, associated with improved neuronal survival and motor performance, and a drastic decrease in mHtt levels. The protective effects of XBP1 deficiency were associated with enhanced macroautophagy in both cellular and animal models of HD. In contrast, ATF4 deficiency did not alter mHtt levels. Although, XBP1 mRNA splicing was observed in the striatum of HD transgenic brains, no changes in the levels of classical ER stress markers were detected in symptomatic animals. At the mechanistic level, we observed that XBP1 deficiency led to augmented expression of Forkhead box O1 (FoxO1), a key transcription factor regulating autophagy in neurons. In agreement with this finding, ectopic expression of FoxO1 enhanced autophagy and mHtt clearance in vitro. Our results provide strong evidence supporting an involvement of XBP1 in HD pathogenesis probably due to an ER stress-independent mechanism involving the control of FoxO1 and autophagy levels.
Most neurodegenerative diseases involve the accumulation of misfolded proteins in the nervous system. Impairment of protein degradation pathways such as autophagy is emerging as a consistent and transversal pathological phenomenon in neurodegenerative diseases, including Alzheimer's, Huntington's, and Parkinson's disease. Genetic inactivation of autophagy in mice has demonstrated a key role of the pathway in maintaining protein homeostasis in the brain, triggering massive neuronal loss and the accumulation of abnormal protein inclusions. However, the mechanism underlying neurodegeneration due to autophagy impairment remains elusive. A paper in Molecular Neurodegeneration from Abeliovich's group now suggests a role for phosphorylation of Tau and the activation of glycogen synthase kinase 3β (GSK3β) in driving neurodegeneration in autophagy-deficient neurons. We discuss the implications of this study for understanding the factors driving neurofibrillary tangle formation in Alzheimer's disease and tauopathies.
See research article http://www.molecularneurodegeneration.com/content/7/1/48
Most intrinsic death signals converge into the activation of pro-apoptotic BCL-2 family members BAX and BAK at the mitochondria, resulting in the release of cytochrome c and apoptosome activation. Chronic endoplasmic reticulum (ER) stress leads to apoptosis through the upregulation of a subset of pro-apoptotic BH3-only proteins, activating BAX and BAK at the mitochondria. Here we provide evidence indicating that the full resistance of BAX and BAK double deficient (DKO) cells to ER stress is reverted by stimulation in combination with mild serum withdrawal. Cell death under these conditions was characterized by the appearance of classical apoptosis markers, caspase-9 activation, release of cytochrome c, and was inhibited by knocking down caspase-9, but insensitive to BCL-XL overexpression. Similarly, the resistance of BIM and PUMA double deficient cells to ER stress was reverted by mild serum withdrawal. Surprisingly, BAX/BAK-independent cell death did not require Cyclophilin D (CypD) expression, an important regulator of the mitochondrial permeability transition pore. Our results suggest the existence of an alternative intrinsic apoptosis pathway emerging from a cross talk between the ER and the mitochondria.
Creutzfeldt-Jakob disease (CJD) is the most frequent human Prion-related disorder (PrD). The detection of 14-3-3 protein in the cerebrospinal fluid (CSF) is used as a molecular diagnostic criterion for patients clinically compatible with CJD. However, there is a pressing need for the identification of new reliable disease biomarkers. The pathological mechanisms leading to accumulation of 14-3-3 protein in CSF are not fully understood, however neuronal loss followed by cell lysis is assumed to cause the increase in 14-3-3 levels, which also occurs in conditions such as brain ischemia. Here we investigated the relation between the levels of 14-3-3 protein, Lactate dehydrogenase (LDH) activity and expression of the prion protein (PrP) in CSF of sporadic and familial CJD cases. Unexpectedly, we found normal levels of LDH activity in CJD cases with moderate levels of 14-3-3 protein. Increased LDH activity was only observed in a percentage of the CSF samples that also exhibited high 14-3-3 levels. Analysis of the PrP expression pattern in CSF revealed a reduction in PrP levels in all CJD cases, as well as marked changes in its glycosylation pattern. PrP present in CSF of CJD cases was sensitive to proteases. The alterations in PrP expression observed in CJD cases were not detected in other pathologies affecting the nervous system, including cases of dementia and tropical spastic paraparesis/HTLV-1 associated myelopathy (HAM/TSP). Time course analysis in several CJD patients revealed that 14-3-3 levels in CSF are dynamic and show a high degree of variability during the end stage of the disease. Post-mortem analysis of brain tissue also indicated that 14-3-3 protein is upregulated in neuronal cells, suggesting that its expression is modulated during the course of the disease. These results suggest that a combined analysis of 14-3-3 and PrP expression pattern in CSF is a reliable biomarker to confirm the clinical diagnosis of CJD patients and follow disease progression.
Thy-1 is a membrane glycoprotein suggested to stabilize or inhibit growth of neuronal processes. However, its precise function has remained obscure, because its endogenous ligand is unknown. We previously showed that Thy-1 binds directly to αVβ3 integrin in trans eliciting responses in astrocytes. Nonetheless, whether αVβ3 integrin might also serve as a Thy-1-ligand triggering a neuronal response has not been explored. Thus, utilizing primary neurons and a neuron-derived cell line CAD, Thy-1-mediated effects of αVβ3 integrin on growth and retraction of neuronal processes were tested. In astrocyte-neuron co-cultures, endogenous αVβ3 integrin restricted neurite outgrowth. Likewise, αVβ3-Fc was sufficient to suppress neurite extension in Thy-1(+), but not in Thy-1(−) CAD cells. In differentiating primary neurons exposed to αVβ3-Fc, fewer and shorter dendrites were detected. This effect was abolished by cleavage of Thy-1 from the neuronal surface using phosphoinositide-specific phospholipase C (PI-PLC). Moreover, αVβ3-Fc also induced retraction of already extended Thy-1(+)-axon-like neurites in differentiated CAD cells as well as of axonal terminals in differentiated primary neurons. Axonal retraction occurred when redistribution and clustering of Thy-1 molecules in the plasma membrane was induced by αVβ3 integrin. Binding of αVβ3-Fc was detected in Thy-1 clusters during axon retraction of primary neurons. Moreover, αVβ3-Fc-induced Thy-1 clustering correlated in time and space with redistribution and inactivation of Src kinase. Thus, our data indicates that αVβ3 integrin is a ligand for Thy-1 that upon binding not only restricts the growth of neurites, but also induces retraction of already existing processes by inducing Thy-1 clustering. We propose that these events participate in bi-directional astrocyte-neuron communication relevant to axonal repair after neuronal damage.
Axonal degeneration is an active process that has been associated with neurodegenerative conditions triggered by mechanical, metabolic, infectious, toxic, hereditary and inflammatory stimuli. This degenerative process can cause permanent loss of function, so it represents a focus for neuroprotective strategies. Several signaling pathways are implicated in axonal degeneration, but identification of an integrative mechanism for this self-destructive process has remained elusive. Here, we show that rapid axonal degeneration triggered by distinct mechanical and toxic insults is dependent on the activation of the mitochondrial permeability transition pore (mPTP). Both pharmacological and genetic targeting of cyclophilin D, a functional component of the mPTP, protects severed axons and vincristine-treated neurons from axonal degeneration in ex vivo and in vitro mouse and rat model systems. These effects were observed in axons from both the peripheral and central nervous system. Our results suggest that the mPTP is a key effector of axonal degeneration, upon which several independent signaling pathways converge. Since axonal and synapse degeneration are increasingly considered early pathological events in neurodegeneration, our work identifies a potential target for therapeutic intervention in a wide variety of conditions that lead to loss of axons and subsequent functional impairment.
Endoplasmic reticulum (ER) stress is a hallmark feature of secretory cells and many diseases including cancer, neurodegeneration, and diabetes. Adaptation to protein folding stress is mediated by the activation of an integrated signal transduction pathway known as the unfolded protein response (UPR). The UPR signals through three distinct stress sensors located at the ER membrane, IRE1α, ATF6 and PERK. Although PERK and IRE1α share functionally similar ER-luminal sensing domains and both are simultaneously activated in cellular paradigms of ER stress in vitro, they are selectively engaged in vivo by the physiological stress of unfolded proteins. The differences in terms of tissue-specific regulation of the UPR may be explained by the formation of distinct regulatory protein complexes. This concept is supported by the recent identification of adaptor and modulator proteins that directly interact with IRE1α. In this review we discuss recent evidence supporting a model where IRE1α signaling emerges as a highly regulated process, controlled by the formation of a dynamic scaffold onto which many regulatory components assemble.
Prion-related disorders (PrDs) are caused by the accumulation of a misfolded and protease-resistant form of the cellular prion, leading to neuronal dysfunction and massive neuronal loss. In humans, PrDs have distinct etiologies including sporadic, infectious and familial forms, which present common clinical features; however, the possible existence of common neuropathogenic events are not known. Several studies suggest that alterations in protein folding and quality control mechanisms at the endoplasmic reticulum (ER) are a common factor involved in PrDs. However, the mechanism underlying ER dysfunction in PrDs remains unknown. We have recently reported that alterations in ER calcium homeostasis are common pathological events observed in both infectious and familial PrD models. Perturbation in calcium homeostasis directly correlated with the occurrence of ER stress and higher susceptibility to protein folding stress. We envision a model where alterations in ER function are central and common events underlying prion pathogenesis, leading to general alterations on protein homeostasis networks.
prion protein; calcium; endoplasmic reticulum stress; unfolded protein response; chaperones
Prion-related disorders (PrDs) are fatal neurodegenerative disorders characterized by progressive neuronal impairment as well as the accumulation of an abnormally folded and protease resistant form of the cellular prion protein, termed PrPRES. Altered endoplasmic reticulum (ER) homeostasis is associated with the occurrence of neurodegeneration in sporadic, infectious and familial forms of PrDs. The ER operates as a major intracellular calcium store, playing a crucial role in pathological events related to neuronal dysfunction and death. Here we investigated the possible impact of PrP misfolding on ER calcium homeostasis in infectious and familial models of PrDs. Neuro2A cells chronically infected with scrapie prions showed decreased ER-calcium content that correlated with a stronger upregulation of UPR-inducible chaperones, and a higher sensitivity to ER stress-induced cell death. Overexpression of the calcium pump SERCA stimulated calcium release and increased the neurotoxicity observed after exposure of cells to brain-derived infectious PrPRES. Furthermore, expression of PrP mutants that cause hereditary Creutzfeldt-Jakob disease or fatal familial insomnia led to accumulation of PrPRES and their partial retention at the ER, associated with a drastic decrease of ER calcium content and higher susceptibility to ER stress. Finally, similar results were observed when a transmembrane form of PrP was expressed, which is proposed as a neurotoxic intermediate. Our results suggest that alterations in calcium homeostasis and increased susceptibility to ER stress are common pathological features of both infectious and familial PrD models.
The cytosolic chaperone Hsp72 directly modulates stress sensing in response to the accumulation of unfolded proteins in the endoplasmic reticulum and promotes cell survival.
Endoplasmic reticulum (ER) stress is a feature of secretory cells and of many diseases including cancer, neurodegeneration, and diabetes. Adaptation to ER stress depends on the activation of a signal transduction pathway known as the unfolded protein response (UPR). Enhanced expression of Hsp72 has been shown to reduce tissue injury in response to stress stimuli and improve cell survival in experimental models of stroke, sepsis, renal failure, and myocardial ischemia. Hsp72 inhibits several features of the intrinsic apoptotic pathway. However, the molecular mechanisms by which Hsp72 expression inhibits ER stress-induced apoptosis are not clearly understood. Here we show that Hsp72 enhances cell survival under ER stress conditions. The UPR signals through the sensor IRE1α, which controls the splicing of the mRNA encoding the transcription factor XBP1. We show that Hsp72 enhances XBP1 mRNA splicing and expression of its target genes, associated with attenuated apoptosis under ER stress conditions. Inhibition of XBP1 mRNA splicing either by dominant negative IRE1α or by knocking down XBP1 specifically abrogated the inhibition of ER stress-induced apoptosis by Hsp72. Regulation of the UPR was associated with the formation of a stable protein complex between Hsp72 and the cytosolic domain of IRE1α. Finally, Hsp72 enhanced the RNase activity of recombinant IRE1α in vitro, suggesting a direct regulation. Our data show that binding of Hsp72 to IRE1α enhances IRE1α/XBP1 signaling at the ER and inhibits ER stress-induced apoptosis. These results provide a physical connection between cytosolic chaperones and the ER stress response.
The endoplasmic reticulum (ER) is responsible for production and folding of secreted proteins. When the protein folding machinery cannot keep up with demand, misfolded proteins accumulate, leading to a state of ER stress that contributes to diseases such as cancer, neurodegeneration, diabetes, and myocardial infarct. The unfolded protein response (UPR) is an intracellular signaling network activated in response to ER stress. It initially tries to restore normal ER homeostasis, but if the damage is too severe cell death pathways mediated by cytosolic and mitochondrial proteins are activated. The molecular mechanisms involved in the transition of the UPR from a protective to an apoptotic phase are unclear. IRE1α is an ER membrane protein that acts as a sensor of ER stress. A number of proteins can interact with IRE1α to regulate its function, which includes an RNase activity responsible for inducing the unconventional splicing of the transcript for a downstream signaling protein called XBP-1. Here, we report that Hsp72, a stress-inducible cytosolic molecular chaperone, can bind to and enhance the RNase activity of IRE1α, providing an important molecular link between the heat shock response and the ER stress response. Importantly, increased production of active XBP-1 was necessary for Hsp72 to exert its prosurvival effect under conditions of ER stress. Our results suggest a mechanism whereby Hsp72 overexpression helps cells adapt to long-term ER stress in vivo by enhancing the pro-survival effects of the IRE1α/XBP1 branch of the UPR.
Adaptation to endoplasmic reticulum (ER) stress depends on the activation of an integrated signal transduction pathway known as the unfolded protein response (UPR). Bax inhibitor-1 (BI-1) is an evolutionarily conserved ER-resident protein that suppresses cell death. Here we have investigated the role of BI-1 in the UPR. BI-1 expression suppressed IRE1α activity in fly and mouse models of ER stress. BI-1 deficient cells displayed hyperactivation of the ER stress sensor IRE1α, leading to increased levels of its downstream target X-Box binding protein-1 (XBP-1) and upregulation of UPR target genes. This phenotype was associated with the formation of a stable protein complex between BI-1 and IRE1α, decreasing its ribonuclease activity. Finally, BI-1 deficiency increased the secretory activity of primary B cells, a phenomenon regulated by XBP-1. Our results suggest a new role for BI-1 in early adaptive responses against ER stress which contrasts with its known downstream function in apoptosis.
Transmissible Spongiform Encephalopathies are fatal and infectious neurodegenerative diseases characterized by extensive neuronal apoptosis and the accumulation of an abnormally folded form of the cellular prion protein (PrP), denoted PrPSC. Compelling evidence suggests the involvement of several signaling pathways in prion pathogenesis, including proteasome dysfunction, alterations in the protein maturation pathways and the unfolded protein response. Recent reports indicate that endoplasmic reticulum stress due to the PrP misfolding may be a critical factor mediating neuronal dysfunction in prion diseases. These findings have applications for developing novel strategies for treatment and early diagnosis of transmissible spongiform encephalopathies and other neurodegenerative diseases.
Prion related disorders; apoptosis; PrPSC; proteasome; ER stress; glucose-regulated proteins; caspase-12; PrPSC-like; aggresomes
Prion diseases are fatal and infectious neurodegenerative disorders characterized by the accumulation of an abnormally folded form of the prion protein (PrP), termed PrPSc. Prion replication triggers endoplasmic reticulum (ER) stress, neuronal dysfunction, and apoptosis. In this study we analyze the effect of perturbations in ER homeostasis on PrP biochemical properties and prion replication. ER stress led to the generation of a misfolded PrP isoform, which is detergent-insoluble and protease-sensitive. To understand the mechanism by which ER stress generates PrP misfolding, we assessed the contribution of different signaling pathways implicated in the unfolded protein response. Expression of a dominant negative form of IRE1α or XBP-1 significantly increased PrP aggregation, whereas overexpression of ATF4 or an active mutant form of XBP-1 and ATF6 had the opposite affect. Analysis of prion replication in vitro revealed that the PrP isoform generated after ER stress is more efficiently converted into PrPSc compared with the protein extracted from untreated cells. These findings indicate that ER-damaged cells might be more susceptible to prion replication. Because PrPSc induces ER stress, our data point to a vicious cycle accelerating prion replication, which may explain the rapid progression of the disease.
The pathogenic mechanism(s) underlying neurodegenerative diseases associated with protein misfolding is unclear. Several studies have implicated ER stress pathways in neurodegenerative conditions, including prion disease, amyotrophic lateral sclerosis, Alzheimer’s disease and many others. The ER stress response and upregulation of ER stress-responsive chaperones is observed in the brains of patients affected with Creutzfeldt-Jacob disease and in mouse models of prion diseases. In particular, the processing of caspase-12, an ER-localized caspase, correlates with neuronal cell death in prion disease. However, the contribution of caspase-12 to neurodegeneration has not been directly addressed in vivo. We confirm that ER stress is induced and that caspase-12 is proteolytically processed in a murine model of infectious prion disease. To address the causality of caspase-12 in mediating infectious prion pathogenesis, we inoculated mice deficient in caspase-12 with prions. The survival, behavior, pathology and accumulation of proteinase K-resistant PrP are indistinguishable between caspase-12 knockout and control mice, suggesting that caspase-12 is not necessary for mediating the neurotoxic effects of prion protein misfolding.
B-cell lymphoma protein 2 (Bcl-2) and Bcl-2-associated X protein (Bax), key antiapoptotic and proapoptotic proteins, respectively, have important roles in acute and chronic models of neurologic disease. Several studies have implicated Bax and Bcl-2 in mediating neurotoxicity in prion diseases. To determine whether diminishing apoptotic cell death is protective in an infectious prion disease model we inoculated mice that either were null for proapoptotic Bax or overexpressed antiapoptotic Bcl-2. Interestingly, genetic manipulation of apoptosis did not lessen the clinical severity of disease. Moreover, some disease parameters, such as behavioral alterations and death, occurred slightly earlier in mice that are null for Bax or overexpress Bcl-2. These results suggest that Bax and Bcl-2 mediated apoptotic pathways are not the major contributing factor to the clinical or pathological features of infectious prion disease.
PrP; home cage; amyloid; cell death; necrosis; transmissible
The pathogenic mechanism(s) underlying neurodegenerative diseases associated with protein misfolding is unclear. Several studies have implicated ER stress pathways in neurodegenerative conditions, including prion disease, amyotrophic lateral sclerosis, Alzheimer's disease and many others. The ER stress response and upregulation of ER stress-responsive chaperones is observed in the brains of patients affected with Creutzfeldt-Jacob disease and in mouse models of prion diseases. In particular, the processing of caspase-12, an ER-localized caspase, correlates with neuronal cell death in prion disease. However, the contribution of caspase-12 to neurodegeneration has not been directly addressed in vivo. We confirm that ER stress is induced and that caspase-12 is proteolytically processed in a murine model of infectious prion disease. To address the causality of caspase-12 in mediating infectious prion pathogenesis, we inoculated mice deficient in caspase-12 with prions. The survival, behavior, pathology and accumulation of proteinase K-resistant PrP are indistinguishable between caspase-12 knockout and control mice, suggesting that caspase-12 is not necessary for mediating the neurotoxic effects of prion protein misfolding.